Stabilized lactoperoxidase and glucose oxidase concentrate

ABSTRACT

A stabilized aqueous enzyme concentrate composition which comprises: 
     a) 1000 to 1800 units/ml of lactoperoxidase; 
     b) 1500 to 2750 units/ml of glucose oxidase; 
     c) 10 to 20% w/v of an alkali metal halide salt; and 
     d) a chelating buffering agent present in an amount such that the pH of the composition is in the range of 5.5 to 6.5.

This is a 371 of PCT Application No. PCT/EP 98/02541, filed on Apr. 24,1998.

This invention relates to a stabilized enzyme concentrate of glucoseoxidase and lactoperoxidase.

W091/11105 discloses anti-microbial compositions which contain iodideand hiocyanate anions, glucose oxidase, D-glucose and lactoperoxidase.These compositions have excellent anti-microbial properties and areeffective against bacteria, yeasts and moulds. It is further disclosedthat the compositions may be provided in concentrated substantiallynon-reacting form. Such concentrated compositions maintain physicalseparation of the glucose oxidase and at least one of its substrates,namely D-glucose, water and oxygen, such that H₂O₂ production issubstantially prevented during storage. It is also disclosed that theconcentrated compositions may incorporate at least one buffering agentto minimize the fall of the pH which may otherwise occur afteractivation of the concentrated composition. However, although a verysuccessful product exists in which D-glucose, sodium thiocyanate andpotassium iodide are provided in a substrate solution and thelactoperoxidase and glucose oxidase are provided in a concentratedenzyme solution, there exists a problem with the enzyme solutionregarding its comparatively short shelf-life which is around twelveweeks at ambient temperature. Therefore, there is a need to provide anenzyme solution with a much longer shelf-life.

It is known that the stabilization of an enzyme in solution isdifficult. W090/05182 describes some of the attempts which have beenmade to overcome this problem, for example the addition of sugars orglycerol to enzyme solutions or by freeze drying. W090/05182 states thatfreeze drying is expensive and often results in denaturation and thendiscloses a method of protecting proteins against denaturation on vacuumor air drying which comprises mixing an aqueous solution of the proteinwith a soluble cationic polyelectrolyte and a cyclic polyol, andremoving the water from the solution.

A method of stabilizing proteins in solution is disclosed in W095/10605in which a protein stabilizer additive which comprises two or more of atris compound of formula I: (HOCH₂)₃—C—R, wherein R is various groupsfor example C₁₋₄ alkyl which may be optionally substituted, apolvelectrolyte; a buffer and one or more further components forexample, divalent metal salts. On page 3 of this document it isdisclosed that “The said further component may be selected from thegroup comprising divalent metal ions, chelators, for example EDTA, EGTAor citrate (not with peroxidases) or polyols”. The statement woulddirect the person skilled in the art, faced with the problem offormulating a stable enzyme concentrate containing lactoperoxidase, awayfrom the use of citrates. It would point him instead to the use ofnon-chelating buffers, for example BIS/TRIS.

It will be appreciated that, given the known difficulties of stabilizingone enzyme in solution, the problem is further compounded when a stablemixture of two enzymes in solution is required since each enzyme has itsown optimum requirements which may not be compatible with the optimumrequirements of the other enzyme. In addition, it is not only requiredthat the enzyme concentrate is chemically stable but it must also bepreserved against microbial attack if it is to be used in anti-microbialcompositions. There may be incompatibility between the agent(s) requiredto produce chemical stability and the agent required to producepreservation.

The present invention provides a stabilized aqueous enzyme concentratecomposition which comprises:

a) 1000 to 1800 units/ml of lactoperoxidase;

b) 1500 to 2750 units/ml of glucose oxidase;

c) 10 to 20% w/v of an alkali metal halide salt; and

d) a chelating buffering agent present in an amount such that the pH ofthe composition is in the range of 5.5 to 6.5.

Although it is known that sodium chloride may be used to preservelactoperoxidase, for example at a level of 1.8% or 12%, it isnevertheless surprising that a concentrate of lactoperoxidase andglucose oxidase may be stabilized chemically and microbially preservedfor long periods by a combination of a chelating buffering agent and analkali metal salt.

WO95/26137 (page 43) discloses the effect of pH on the anti-microbialactivity of final compositions which have been prepared by combining anddiluting two concentrates (Phase A and Phase B) to 0.9% and 0.05%.

Component Concentration Phase A (w/v %) D-glucose 45 to 55 Sodiumthiocyanate 0.42 to 0.52 Potassium iodide 0.66 to 0.80 Phase BLactoperoxidase 5,500 Units/ml Glucose oxidase 2,250 Units/ml

The pH of each phase was adjusted to between 5.5 and 6.5 with buffersolutions. The pH of each combined concentrate mixture after dilutionwas adjusted using a citrate/phosphate buffer. However, this documentdoes not disclose or suggest the unexpected and beneficial effect ofobtaining an enzyme concentrate which is stable over a prolonged period.

EP 307,376 and WO91/11105 disclose diluted anti-microbial compositionswhich contain citrate salts and/or sodium chloride. However, there is nodisclosure of a stabilised enzyme concentrate according to the presentinvention.

EP 252,051 discloses a method of stabilizing lactoperoxidase in milkproducts, foodstuffs and pharmaceuticals to which the enzymelactoperoxidase has been added, wherein the product containinglactoperoxidase is adjusted with regard to pH, so that the pH is in therange 3.25 to 6 at the dissolution in water. It does not disdose astabilised enzyme concentrate according to the present invention.

Suitably, the enzyme concentrate is stable for at least 6 months.Preferably the enzyme concentrate is stable for at least 9 months. Morepreferably the enzyme concentrate is stable for at least 12 months. Mostpreferably the enzyme concentrate is stable for at least 18 months.

The stability of the enzyme concentrate is determined by assaying theactivity of the lactoperoxidase and glucose oxidase at regular intervalsusing standard assay techniques which are given in the examples of thisspecification. The efficacy of antimicrobial preservation is assessedusing the British Pharmacopeia test as given in the 1993 volume,Appendix XVIC A191.

Suitably the stability of the enzyme concentrate is such that theactivities of lactoperoxidase and glucose oxidase are maintained at atleast 60% of their original activity over the time period examined.Preferably the activities of lactoperoxidase and glucose oxidase aremaintained at least 75% of their original activity after storage for 6months at 25° C. More preferably the activities of lactoperoxidase andglucose oxidase are maintained at least 75% of their original activityafter storage for 12 months at 25° C.

Preferably the alkali metal halide is selected from sodium chloride,potassium chloride, sodium bromide, potassium bromide, sodium iodide orpotassium iodide or mixtures thereof. More preferably the alkali metalhalide is sodium chloride.

Preferably the concentration of the alkali metal halide is in the rangeof 12 to 18% w/v. More preferably the concentration of the alkali metalhalide is in the range of 14 to 16% w/v. Most preferably theconcentration of the alkali metal halide is in the range of 14.5 to15.5% w/v.

Suitably the chelating buffering agent is selected from one or more ofthe following: alkali metal salts of citric, phthalic, tartaric, adipicor succinic acid. Preferably the chelating buffering agent is trisodiumcitrate. Suitably the amount of chelating buffering agent present isthat required to give a final pH of in the range 5.5 to 6.5. Preferablythe amount of buffering agent is such that the pH of the composition isin the range 5.7 to 6.2. Most preferably the amount of buffering agentpresent is such that the pH of the composition is in the range of 5.9 to6.1. Optionally monosodium orthophosphate may be present in an amountranging from 0.05 M to 0.5 M, preferably 0.25 M. Preferably the weightof trisodium citrate used is in the range 0.5 to 1.5% w/v.

The invention will be understood with reference to the followingnon-limiting tests and examples.

Effect of Buffer

Five different solutions of lactoperoxidase and Glucox PS were examinedin this study:

a) Standard Enzyme Concentrate Solution

b) Lactoperoxidase and Glucox PS in water at pH 6.0

c) Lactoperoxidase and Glucox PS in 50 mM citrate buffer pH 6.0

d) Lactoperoxidase and Glucox PS in 50 mM BIS-TRIS buffer pH 6.0

e) Lactoperoxidase and Glucox PS in 50 mM BIS-TRIS buffer pH 7.0

Glucose PS is a freeze dried preparation of Glucose Oxidase Solution a)Lactoperoxidase 1000-1800 U/ml Glucose oxidase 1500-2750 U/ml pH  5.5 to6.5 Wt/ml at 20° C. 1.00 to 1.02 g.

Solutions b) to e) were made up as follows.

A mixture of 1.6 1 of lactoperoxidase (2800 units/ml) and Glucox PS(4250 units/ml) in water for injection (WFI) was made up.${{{{The}\quad {weight}\quad {of}\quad {lactoperoxidase}\quad {required}\quad (g)} = \frac{2800 \times 1600}{{Activity}\quad \left( {{units}/{mg}} \right) \times 1000}}{The}\quad {weight}\quad {of}\quad {Glucox}\quad {PS}\quad {required}\quad (g)} = \frac{4250 \times 1600}{{Activity}\quad \left( {{units}/{mg}} \right) \times 1000}$

The above solution was divided into 4 equal parts (i to iv) and thefollowing materials were added.

i) nothing

ii) 8.405 g citric acid

iii) 8.368 g BIS/TRIS

iv) 8.368 g BIS/TRIS

The volume of the solutions i to iv were made up to 750 ml with WFI.

The pH of each of the above solutions was adjusted using 1 M NaOH or 1 MHCI as appropriate, to:

i) 5.9to6.1

ii) 5.9to6.1

iii) 5.9 to 6.1

iv) 6.9 to 7.1

The volume of the solutions i to iv were made up to 800 ml with WFI.

All five solutions were divided into 25×30 ml samples. One sample ofeach was retained for analysis as the initial sample and eight samplesof each were stored at 4° C., 25° C. and 40° C.

The solutions were assayed as described in the table below by the assaymethods for glucose oxidase and lactoperoxidase which are describedlater.

Temp 3 1 (° C.) Initial days week 2 weeks 1 month 2 months 4 months  4 —L L L L 25 L, G, L, G, pH L, pH L, G, pH pH 40 — L L L L L L =lactoperoxidase activity G = glucose oxidase activity B = pH (afterother assays have been successfully completed)

The results are shown in Tables 1, 2 and 3 and FIG. 1.

TABLE 1 Glucose Oxidase Activities (units/ml) 91 Days Initial 28 Days 4°C. 25° C. 40° C. Control 1778 2166 1922 1561 103 WFI 1867 2110 ndCitrate 1532 1956 1611 1617 5.5 Bis-Tris pH 6.0 1814 1568 nd Bis-Tris pH7.0 1957 1754 nd nd = not done

TABLE 2 Lactoperoxidase Activities (units/ml) 3 7 14 Sample Initial DaysDays Days 28 Days 55 Days 91 Days Con- 4° C. 1258 1325 1559 1713 trol25° C. 1305 1070 1265 1031 935 40° C. 1051 734 737 516 502 733 WFI 4° C.1173 1323 1289 25° C. 1237 1103 1134 872 40° C. 951 1028 956 328 572 Ci-4° C. 974 1130 1199 1417 trate 25° C. 1100 1031 1077 1156 1104 40° C.1040 997 1137 930 973 1092 Bis- 4° C. 1153 1274 1374 Tris 25° C. 1163839 665 359 pH 40° C. 869 446 233 168 30.2 6.0 Bis- 4° C. 1192 1301 1378Tris 25° C. 1257 1147 1174 1017 pH 40° C. 1071 962 845 637 545 7.0

TABLE 3 pH of Samples of Lactoperoxidase and Glucose Oxidase Stored atVarious Temperatures Sample Initial 14 Days 28 Days 55 Days 91 DaysControl  4° C. nd 6.08 6.08 6.10 6.10 25° C. nd 6.04 6.07 6.06 6.07 40°C. nd 6.06 6.04 6.02 5.99 WFI  4° C. nd 5.89 6.03 6.14 nd 25° C. 6.006.13 6.23 6.40 nd 40° C. nd 6.10 6.32 6.39 nd Citrate  4° C. nd 6.046.01 6.03 6.05 25° C. 6.00 6.01 6.05 6.16 6.42 40° C. nd 6.07 6.08 6.116.17 Bis-Tris pH 6.0  4° C. nd 5.84 5.98 5.94 nd 25° C. 6.00 5.59 5.645.36 nd 40° C. nd 5.26 5.30 5.00 nd Bis-Tris pH 7.0  4° C. nd 6.97 7.037.02 nd 25° C. 7.00 6.87 6.93 6.91 nd 40° C. nd 6.83 6.90 6.81 nd

Effect of Buffer and Alkali Metal Salt

A citrate buffer was prepared by dissolving citric acid monohydrate(52.53 g) in approximately 3.5 l water for injection (WFI). The buffersolution was split into 5 equal aliquots of 700 ml. Sodium chloride (150g) was added to the first aliquot and the pH of this test solution wasadjusted to between 5.9 and 6.1 with 6 M NaOH. Sufficientlactoperoxidase and glucose oxidase were added to give a test solutioncontaining 1500 U/ml lactoperoxidase and 2125 U/ml glucose oxidase.${{Weight}\quad {of}\quad {lactoperoxidase}\quad (g)} = \frac{1500 \times 1000}{{activity} \times 1000}$${{Weight}\quad {of}\quad {glucose}\quad {oxidase}\quad (g)} = \frac{2125 \times 1000}{{activity} \times 1000}$

The pH of the test solution was adjusted to between 5.9 and 6.1 with 6 MNaOH. The volume of the test solution was made up to 1.0 l with WFI.

100 ml of the test solution was submitted for BP challenge testing andthe remainder of the test solution was split into 19 samples of 40 ml.

The samples were stored and assayed as indicated in the table below.

Temp Time (months) (° C.) 0 0.5 1 2 3 4 5 6 9 12  4 L L L L 25 L, G, B LL L L, G L L L, G L, G L, G 40 L L L L, G L, G L = lactoperoxidase assayG = glucose oxidase assay B = BP challenge

The results are given in Tables A, B, C, D and E. The results confirmthat the activity of both enzymes was maintained.

ENZYMATIC ASSAY OF LACTOPEROXIDASE

All reagents and sample solutions were prepared fresh daily.

Purified water. HPLC grade as supplied by Romil Chemicals was suitable.

Phosphate Buffer (0.1 M pH 5.5)

(a) Potassium dihydrogen phosphate (1.361 g) was weighed into a 100 mlgraduated flask, dissolved in and diluted to volume with purified water.

(b) Dipotassium hydrogen phosphate trihydrate (2.282 g) was weighed intoa 100 ml graduated flask, dissolved in and diluted to volume withpurified water.

To 50 ml of solution (a) was added sufficient solution (b) to give pH5.5.

ABTS Solution

ABTS solution (100 mM) was prepared by dissolving2,2′-Azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt(ABTS) (0.55 g) in 10 ml of purified water.

Hydrogen Peroxide Solution

0.1 ml of 30% hydrogen peroxide solution was dissolved in 120 ml ofpurified water. The absorbance of this solution was measured at 240 nmin 1-cm cells and adjusted if necessary by further additions of hydrogenperoxide or purified water to give an absorbance reading of about 0.40.

Dipotassium Hydrogen Phosphate Solution (0.2 M)

Dipotassium hydrogen phosphate trihydrate (22.820 g) was dissolved in500 ml of purified water.

Strong Sample Solution

1.0 ml of the sample was pipetted into a 200 ml graduated flask anddiluted to volume with 0.2 M dipotassium hydrogen phosphate solution.

Dilute Sample Solution

1.0 ml of the strong sample solution was pipetted into a 200 mlgraduated flask and diluted to volume with 0.2 M dipotassium hydrogenphosphate solution.

Procedure

2.20 ml of phosphate buffer, 0.70 ml of ABTS solution and 0.10 ml ofhydrogen peroxide solution were pipetted into a 1 cm silica or glasscuvette and mixed. The sample was equilibrated in a water bath at 25° C.then 0.020 ml of dilute sample solution was added. The sample was mixedand again equilibrated at 25° C. for 30 seconds. The cell wasimmediately transferred to the spectrophotometer and the change inabsorbance was measured over a period of 2 minutes at a wavelength of436 nm. The change in absorbance per minute was calculated. The aboveprocedure was carried out in triplicate and the mean change inabsorbance per minute (−A/min) for the three determinations wascalculated.${{Then}:{{lactoperoxidase}\quad \left( {{units}/{ml}} \right)}} = {\frac{3.02}{29.3} \times \frac{200}{1} \times \frac{5}{1} \times \frac{{- A}/\min}{0.02}}$

ENZYMATIC ASSAY OF GLUCOSE OXIDASE

All reagents and sample solutions were prepared fresh daily.

Purified water. HPLC grade as supplied by Romil Chemicals was suitable.

Citric Buffer pH5

Citric acid monohydrate (10.08 g) was weighed into a 500 ml graduatedflask, 100 ml of M sodium hydroxide solution was added and the solutiondiluted to volume with purified water.

Benzoquinone Solution

Benzoquinone (puriss) (0.05 g) was weighted into a 50 ml graduatedflask, dissolved in and diluted to volume with purified water.

Glucose Solution

Anhydrous D-glucose (18.0 g) was weighed into a 100 ml graduated flask,dissolved in and diluted to volume with purified water. The solution wasallowed to stand for 24 hours before use.

Strong Sample Solution

5.0 ml of the sample solution was pipetted into a 100 ml graduated flaskand diluted to volume with citrate buffer.

Dilute Sample Solution

5.0 ml of strong sample solution was pipetted into a 100 ml graduatedflask and diluted to volume with citrate buffer.

Benzoquinone solution (2.0 ml) and 1.0 ml of glucose solution werepipetted into each of two 1 cm silica cuvettes. Into one cell 1.0 ml ofcitrate buffer was pipetted. The solution was mixed by inversion andplaced in the reference cell compartment of the spectrophotometer. Intothe other cell was pipetted 0.95 ml of citrate buffer. The solution wasmixed and allowed to equilibrate at 25° C. in a water bath. The cell wasplaced in the sample compartment of the spectrophotometer and theinstrument was zeroed. 0.05 ml of dilute sample solution was added tothe sample cell. The sample was mixed by inversion and the change inabsorbance was immediately recorded over a period of 1-2 minutes at awavelength of 290 nm. The change in absorbance per minute wascalculated. The above procedure was carried out in triplicate and themean change in absorbance per minute (−A/min) for the threedeterminations was calculated.${{Then}:{{glucose}\quad {oxidase}\quad \left( {{units}/{ml}} \right)}} = {\frac{{- A}/\min}{2.31} \times \frac{4.0}{0.05} \times \frac{100}{5} \times \frac{100}{5}}$

BP Test

The test for the efficacy of Antimicrobial Preservation was carried outusing a modification of the method described in the British Pharmacopeia1993 Appendix XVIC A191 using the following test organisms:

Aspergillus niger IMI 149 007

Candida albicans (ATCC 10231)

Pseudomonas aeriginosa (NCIMB 8626)

Staphylococcus aureus (NCIMB 9518).

Saccharomyces cerevisiae (NYC 87)

mixed bacterial inoculum based on:

S. aureus, S. epidermidis and Streptococcus haemolyticus.

These were used at two challenge levels, namely 10⁶ per ml and 10² perml in separate tests.

The criteria of acceptance used, were as given in the 1995 addendum tothe British Pharmacopeia in Appendix XVII F A407 and are shown below:

Log reduction 2d 7d 14d 28d Bacteria A 2 3 — NI B — — 3 NI Fungi A — — 2NI B — — 1 NI The A criteria express the recommended efficacy to beachieved. In justified cases where the A criteria cannot be attained,for example, for reasons of an increased risk of adverse reactions, theB criteria must be satisfied. NI: No increase; d = days.

Due to intended use of the stabilised enzyme solution and the low riskof contamination and spoilage after manufacture, it was deemedappropriate to modify the above criteria. The pass requirements werebased on the B criteria with the exception that absence of growth forthe Gram positive bacteria after inoculation was judged to beacceptable.

The stabilised described previously was tested both initially and after6 months storage and was found to be acceptably preserved when submittedto the above modified test.

TABLE A Lactoperoxidase Activity U/ml versus time stored at 4° C.Activity TIME (months) (U/ml) 0 1 3 6 12 Dil 1 1463.6 1379.8 1619.41691.1 1163 1566.1 1327.2 1726.3 1324.5 1432 1515.8 1380.6 1668.6 1495.5 655 Mean 1515.0 1362.5 1083 Dil 2 1403.0 1244.9 1776.4 1140.4  9181743.9 1508.5 1952.3 1733.9 1022 1480.9 1915.2 1587.4 1207 1444.9 Mean1518.2 1376.7 1049 Combined Mean 1516.6 1369.6 1776.2 1495.4 1066

TABLE B Lactoperoxidase Activity U/ml versus time stored at 25° C.Activity TIME (months) (U/ml) 0 0.5 1 2 3 4 5 6 12 Dil 1 1463.6 1547.6954.6 1541.7 2832.6 1738.1 2033.4 892.0 986 1566.1 1509.2 843.2 1429.62382.0 1354.3 1787.2 1241.7 1110 1515.8 1583.5 971.6 1417.1 2079.72100.6 1795.4 1816.9 1057 1536.3 1051 Mean 1515.0 1546.8 923.2 1731.01872.0 1950 Dil 2 1403.0 1351.5 1070.6 1813.1 2067.8 2244.8 1766.51772.6 1983 1743.9 1517.1 1037.2 1734.7 2209.4 2137.5 2058.3 1883.9 16871480.9 955.5 1926.3 1915.9 2126.6 2050.2 1702.1 1444.9 1621.4 1873 Mean1518.2 1496.6 1021.2 2169.6 1958.3 1462 Combined 1516.6 1521.7 972.21628.4 2247.9 1870.2 1915.1 1551.5 1706 Mean

TABLE C Lactoperoxidase Activity U/ml versus time stored at 4° C.Activity TIME (months) (U/ml) 0 0.5 1 2 3 6 Dil 1 1463.6 2423.2 1255.11386.5 1885.9 2125.2 1566.1 2455.4 1045.0 1398.8 1635.3 2049.8 1515.81941.2 1168.0 1502.7 1645.9 2232.6 Mean 1515.0 2273.2 1156.0 Dil 21403.0 2452.9 1103.6 1434.4 2333.8 1990.9 1743.9 1970.6  975.2 1702.31027.4 1937.9 1480.9 2107.1 1002.7 1529.3 1789.6 1444.9 1465.7 Mean1518.2 1999.0 1027.2 Combined 1516.6 2136.1 1091.6 1484.9 1676.2 2021.0Mean

TABLE D Glucose Oxidase Activity in U/ml versus time of storage at 25°C. Activity TIME (months) (U/ml) 0 2 3 6 12 Dil 1 1673.8 1698.8 2234.62265.7 1349 1554.3 1762.7 2007.9 2135.5 1675 1534.9 1689.0 2076.3 2133.11583 1268.6 Mean 1507.9 1535 Dil 2 1705.3 1553.6 1685.9 2258.3 11471457.6 1805.6 1675.3 2201.4 1451 1492.3 1748.1 1673.6 2194.8 1635 Mean1551.7 1411 Combined Mean 1529.8 1710.0 1892.2 2198.1 1473

TABLE E Glucose Oxidase Activity in U/ml versus time of storage at 25°C. Activity TIME (months) (U/ml) 0 2 3 6 Dil 1 1673.8 1217.8 957.5 483.81554.3 1206.5 947.7 514.8 1534.9 1174.9 881.6 463.7 1268.6 Mean 1507.9Dil 2 1705.3 1142.8 1095.3  444.7 1457.6 1133.9 1073.8  440.5 1492.31159.6 991.7 427.8 Mean 1551.7 Combined Mean 1529.8 1173.0 991.3 462.5

What is claimed is:
 1. A stabilized aqueous enzyme concentratecomposition which comprises: a) 1000 to 1800 units/ml oflactoperoxidase; b) 1500 to 2750 units/ml of glucose oxidase; c) 10 to20% w/v of an alkali metal halide salt; and d) a chelating bufferingagent present in an amount such that the pH of the composition is in therange of 5.5 to 6.5.
 2. A composition as claimed in claim 1 which isstable for at least 6 months.
 3. A composition as claimed in claim 1wherein the activities of lactoperoxidase and glucose oxidase aremaintained at at least 75% of their original activity after storage for6 months at 25° C.
 4. A composition as claimed in claim 1 wherein thealkali metal salt is sodium chloride.
 5. A composition as claimed inclaim 4 wherein concentration of sodium chloride is 14 to 16% w/v.
 6. Acomposition as claimed in claim 1 in which the buffering agent is asodium citrate.
 7. A composition as claimed in claim 6 in which saidsodium citrate is trisodium citrate which is present in an amount in therange 0.5 to 1.5% w/v.
 8. A composition as claimed in claim 6 which alsocontains monosodium orthophosphate in an amount ranging from 0.05 M to0.5 M.